1. Field of the Invention
This invention herein relates to polymer protein compounds or protein polymer conjugates, a method of making such compounds and a method of stabilizing proteins in the form of the polymer protein conjugates of the invention in a hostile environment.
Proteins are used in a variety of applications. Whether the proteins, such as enzymes, antibodies, and the like, are used in diagnostics, the food industry, the bioconversion of organic compounds in either water or organic solutions etc., a common problem with using proteins in all of these applications is the instability of the protein itself. Enzymes, antibodies, and the like would be more useful if they were stable in a soluble or an insoluble form at elevated temperatures, in non-optimal aqueous solutions or in organic solutions while retaining the ability to recognize and bind molecules, and in some cases, exhibit catalytic activity. The protection of proteins against proteolytic attack is also highly desirable.
Proteins including enzymes are quite sensitive to their environment. Such environmental conditions include the pH of the solution, temperature, shear forces, the presence of organic solvents, buffer conditions, reactions with irreversible and reversible inhibitors, proteolysis of the enzyme by proteins in the solution, adhesion to other proteins or to the wall of the vessel. The stability of a protein is in reference to its conformational stability and biological activity. The effect of temperature, organic solvent, pH, and storage on the stability of the protein is largely determined by the conformational stability of the protein. Inactivation of the protein is primarily due to denaturation of the protein; however, changes in the protein (such as chemical modification) can also occur, resulting in inactivation without denaturation of the protein. When conditions are not optimal, the protein may be partially or fully denatured which results in a decrease or loss of activity.
It is highly desirable to stabilize proteins and their activity because of their potential uses in organic synthesis and diagnostics in the pharmaceutical and agricultural industries. Available methods, however, do not provide a general approach to solving the problem of protein stability. Moreover, the majority of these methods are not applicable where it is advantageous to use the protein or enzyme either in solution or in an insoluble form.
Inactivation and denaturation is of particular concern in the diagnostics field, because the use of proteins such as labile enzymes and antibodies can result in unreliable test results and limited shelf life of a test kit product. Furthermore, the methods of controlling the denaturation of proteins are expensive and burdensome. The effect of immobilization on the stability of enzymes is highly variable. The immobilization can give enzymes that have enhanced, diminished, or unchanged activity relative to the native enzymes. Examples of each effect exist (For a review of the effect of immobilization on the stability of enzymes, see: Zaborsky Immobilized Enzymes CRC Press: Boca Raton at 49-165 (1978)). For example, one possible way to stabilize the structure and reactivity of a protein such as an enzyme is by immobilization on a soluble or insoluble support. In addition to the possible stabilization of the protein, immobilization on insoluble supports is desirable because of the ease of recovery of the protein and the prevalent use of equipment for applications which use insoluble material.
Advantages of immobilization of proteins on a soluble support include higher rates of transfer of substrate to the protein and greater accessibility of the substrates to the protein. Another advantage to the use of a soluble support is that the solubility of the system allows the use of reactor configurations such as ultrafiltration reactors or membrane reactors and use in diagnostic assays where soluble proteins may be necessary. The inconvenience of proteins immobilized on solid supports may also cause problems in assays where optical and electrical measurements are being taken.
Prior to the invention, difficulties associated with the immobilization of proteins including enzymes using known methodologies include: necessary prior activation of the polymeric support, relatively harsh chemical treatments, which destroy or reduce the activity of the protein, necessary for the immobilization process, and the number of functional groups, or the density of functional groups available for the attachment on the polymeric support. The methods do not offer a general solution to the stabilization and immobilization of protein structure.
Moreover, when enzymes and antibodies are used in applications such as an enzyme-linked immunosorbent assay (ELISA), the problem prior to the invention herein was a lack of stability (because of the pH of the solution, temperature, shear forces, the presence of organic solvents, the lack of buffer, reaction with irreversible inhibitors, proteolysis of the antibody and enzyme by proteinases in the solution, and the like) of both the enzymes and the antibodies used. In addition, the sensitivity of the enzymes to organic solutions made the tests limited to aqueous solutions.
Therefore, it is an object of the invention to provide stabilized proteins through polymer protein compounds.
Another object of this invention is to provide a method for stabilizing a protein in an environment which would reduce an intended functional property of the protein.
Still another object of this invention is to provide a method for making the polymer protein compounds of the invention.
Another object of the invention is to provide for a method of synthesizing peptides.
Still another object of the invention is to provide for a method of assaying using the polymer protein compounds of the invention.
These and other objects and advantages of the invention will be found by reference to the following description.